Modeling correlated main-chain motions in proteins for flexible molecular recognition

We describe a new method for modeling protein and ligand main-chain flexibility, and show its ability to model flexible molecular recognition. The goal is to sample the full conformational space, including large-scale motions that typically cannot be reached in molecular dynamics simulations due to the computational intensity, as well as conformations that have not been observed yet by crystallography or NMR. A secondary goal is to assess the degree of flexibility consistent with protein-ligand recognition. Flexibility analysis of the target protein is performed using the graph-theoretic algorithm FIRST, which also identifies coupled networks of covalent and noncovalent bonds within the protein. The available conformations of the flexible regions are then explored with ROCK by random-walk sampling of the rotatable bonds. ROCK explores correlated motions by only sampling dihedral angles that preserve the coupled bond networks in the protein and generates conformers with good stereochemistry, without using a computationally expensive potential function. A representative set of the conformational ensemble generated this way can be used as targets for docking with SLIDE, which handles the flexibility of protein and ligand side-chains. The realism of this protein main-chain conformational sampling is assessed by comparison with time-resolved NMR studies of cyclophilin A motions. ROCK is also effective for modeling the flexibility of large cyclic and polycyclic ligands, as demonstrated for cyclosporin and zearalenol. The use of this combined approach to perform docking with main-chain flexibility is illustrated for the cyclophilin A-cyclosporin complex and the estrogen receptor in complex with zearalenol, while addressing the question of how much flexibility is allowed without hindering molecular recognition.     

Identification of four allelic variants of the dog<Emphasis Type="Italic"> IGHA</Emphasis> gene

Multiple IgA subclasses have been identified in humans, primates and lagomorphs, whereas in mice, cattle and dogs only a single subclass has been identified. The two human subclasses (IgA₁ and IgA₂) are defined by a difference in the length of the hinge region of the chains between the CH₁ and CH₂ domains. The single IgA subclass so far identified in dogs has an -chain hinge region with a predicted amino-acid sequence similar to that of the human ₁ chain. Allelic variants that differ in the coding sequence of the hinge region have been identified in mice and pigs. In order to investigate whether allelic variants are present in dogs, a portion of the IGHA gene from eight individual dogs was cloned and sequenced. Four sequence variants were identified, and these differed in the coding region of their hinge. A major difference between the variants was the presence of a base polymorphism in the splice acceptor site for the second exon, which resulted in shortening of the hinge in two of the variants. Individuals expressed one or two of the variants identified, suggesting they may be heterozygous or homozygous. Further work is required to determine the effect of the variation on the biological activity of dog IgA and any relationship to susceptibility to mucosal disease.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AY576788–AY576795     

Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus-oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6h. After 6h, the oocytes from each group were cultured in embryo culture medium for another 6h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1 +/- 1.7, 46.8 +/- 2.8, 62.8 +/- 3.8 and 139.5 +/- 6.1 for 10, 30, 60 min and 6h, respectively, P < 0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0.04) as the coincubation time was increased (26.6 +/- 2.9%, 29.0 +/- 4.4%, 39.5 +/- 6.2%, and 49.3 +/- 3.0% for 10, 30, 60 min and 6h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm-oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.     

Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.     

Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.     

Two Rice GRAS Family Genes Responsive to N-Acetylchitooligosaccharide Elicitor are Induced by Phytoactive Gibberellins: Evidence for Cross-Talk Between Elicitor and Gibberellin Signaling in Rice Cells

In this study, we present data showing that two members of the GRAS family of genes from rice, CIGR1 and CIGR2 (chitin-inducible gibberellin-responsive), inducible by the potent elicitor N -acetylchitooligosaccharide (GN), are rapidly induced by exogenous gibberellins. The pattern of mRNA accumulation was dependent on the dose and biological activity of the gibberellins, suggesting that the induction of the genes by gibberellin is mediated by a biological receptor capable of specific recognition and signal transduction upon perception of the phytoactive compounds. Further pharmacological analysis revealed that the CIGR1 and CIGR2 mRNA accumulation by treatment with gibberellin is dependent upon protein phosphorylation/dephosphorylation events. In rice calli derived from slender rice 1, a constitutive gibberellin-responsive mutant, or d1, a mutant deficient in the alpha -subunit of the heterotrimeric G-protein, CIGR1 and CIGR2 were induced by a GN elicitor, yet not by gibberellin. Neither gibberellin nor GN showed related activities in defense or development, respectively. These results strongly suggested that the signal transduction cascade from gibberellin is independent of that from GN, and further implied that CIGR1 and CIGR2 have dual, distinct roles in defense and development.     

A theoretical investigation of the evolution and maintenance of mirror-image flowers

Enantiostyly is the deflection of the female sex organ either to the left or to the right side of the floral axis, resulting in mirror-image flowers. Two types of enantiostyly occur: in monomorphic enantiostyly, individuals exhibit both flower forms, whereas in dimorphic enantiostyly, the forms occur on separate plants. Monomorphic enantiostyly is known from at least 10 families, whereas dimorphic enantiostyly is reported in only three. Phylogenetic evidence suggests that monomorphic enantiostyly has evolved from a straight-styled ancestor and that dimorphic enantiostyly is derived from monomorphic enantiostyly. Here, we use theoretical models to investigate the role of pollen transfer in influencing these evolutionary transitions. We used numerical calculations to examine the evolution of monomorphic and dimorphic enantiostyly under different conditions of pollen transfer, inbreeding depression, and pollinator visitation. Our results demonstrate that in comparison to a putative straight-styled ancestor, both monomorphic and dimorphic enantiostyly function to reduce geitonogamous pollen transfer with a concomitant increase in pollen export. Our calculations suggest that the first stage in the evolution of monomorphic enantiostyly involves the deflection of the style only, followed by selection for reciprocity in anther position to promote more precise cross-pollination. Constraints associated with the developmental genetics of left-right asymmetries may account for the low number of evolutionary transitions from monomorphic to dimorphic enantiostyly, despite the evolutionary stability of this condition once it arises.     

Neophilia, innovation and social learning: a study of intergeneric differences in callitrichid monkeys

In a comparative study of neophilia, innovation and social attentiveness we exposed individuals in seven callitrichid species, from three genera, to novel extractive foraging tasks. The results revealed consistently shorter response latencies, higher levels of successful and unsuccessful manipulation, and greater attentiveness to the task and to conspecifics inLeontopithecus (lion tamarins) than in both Saguinus (tamarins) and Callithrix (marmosets). This is consistent with the hypothesis that species dependent upon manipulative and explorative foraging tend to be less neophobic and more innovative than other species. Furthermore, Callithrix appeared to be less neophobic than Saguinus; ifCallithrix is regarded as the greater specialist, this result is inconsistent with the hypothesis that neophobia is associated with foraging specialization. We consider the relevance of our findings to taxonomic relationships, and to technical and Machiavellian intelligence hypotheses and discuss the implications for captive breeding and reintroduction strategies.Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.